DEMONSTRATION OF POLYRIBOSOMES AFTER FERTILIZATION OF THE SEA URCHIN EGG

Fertilization of sea urchin eggs initiates an increased amino acid incorporation into proteins.1' 2 This stimulation of protein synthesis has been attributed to activation of ribosomes.3 Nemer4' I and Wilt and Hultin' found that the synthetic messenger RNA polyuridylic acid stimulates homogenates from unfertilized eggs to synthesize polyphenylalanine. After fertilization, messenger RNA may combine with ribosomes to form polyribosomes. 7I Monroy and Tyler7 recently reported the presence of polyribosomes in fertilized sea urchin eggs. This paper reports the presence of active polyribosomes, some of which are bound to membranes, in fertilized sea urchin eggs prior to the first mitotic division. The relative proportion of membrane-bound polyribosomes, suggested to be the major site of protein synthesis,8 and of free polyribosomes is also presented. Materials and Methods.-Eggs of Lytechinus variegatus were washed three times and resuspended in sterile sea water at 22-240C and fertilized. Thirty minutes after fertilization either a carbon-14-labeled hydrolyzate of algal proteins, or a mixture of nine uniformly labeled amino acids (L-leucine, L-isoleucine, L-valine, I-glutamic acid, L-phenylalanine, L-serine, L-tryptophane, L-aspartic acid, and L-alanine) was added to equal samples of fertilized eggs and unfertilized eggs. Five minutes later the cells were washed three times in a cold isotonic NaCl: KCl solution,9 washed and resuspended in "homogenizing medium," and then gently homogenized by three strokes of a Teflon pestle in a Duall tissue homogenizer. The homogenizing medium contained 0.01 M MgCl2, 0.24 M KCl, and 0.01 M Tris-HCl at pH 7.6, and contained 1 mg/ml of washed bentonite, unless the supernatant was to be treated with ribonuclease. After centrifugation at 10,000 X g at 50C, the supernatant was collected, treated at 4°C for 20 min with 5 ,g/ml of ribonuclease'0 or 0.3 per cent deoxycholatell (depending upon the experiment), and layered on sucrose gradients. One ml of the 10,000 X g supernatant was layered on top of each sucrose gradient made up with a bentonite-treated homogenizing medium. The sucrose gradients were spun for 2 hr at 25,000 rpm on the SW 25 rotor of the Spinco Model L centrifuge at 0-4°C. Fractions were collected for optical density reading (260 mnu), and were then precipitated with 5 per cent trichloroacetic acid in the presence of added carrier albumin (250 ,ug/ml). Each precipitate was collected on a Whatman GF/C filter, and was washed three times with 5 per cent trichloroacetic acid and three times with ethanol. The filters were dried, and the radioactivity was determined in a Nuclear-Chicago gas flow counter. Results.-Early experiments'2 using a linear 15-30 per cent sucrose gradient showed that protein synthesis occurs on polyribosomes in fertilized eggs. However, ribonuclease treatment of the supernatant from fertilized eggs shifted more radioactivity to the ribosome fractions than could be accounted for by loss from the